Using Bacteriophages to Control Salmonella

By Annel K. Greene, PhD, Professor and Center Director
Clemson University Animal Co-Products Research and Education Center

Dr. Xiuping Jiang, professor in the Clemson University Department of Food, Nutrition, and Packaging Sciences, and PhD student Chao “James” Gong recently completed a pilot plant study to test use of bacteriophages to control Salmonella in rendering processing plant environments.

Bacteriophages are tiny viruses that infect only specific bacteria. They are harmless to humans, animals, and plants and cannot be transformed into any form of virus capable of affecting anything other than a bacterium. Ever present in the air and environment around us, it is estimated that more than 10 nonillion bacteriophages exist on Earth.

A bacteriophage is specific for a particular bacterium that carries a matching receptor site. Once a bacteriophage finds a suitable host bacterium, it attaches to the bacterial cell wall and injects its viral genetic codes into the bacterium. The viral genetic codes quickly take over the machinery of the bacterium and force the bacterium to generate multiple copies of the bacteriophage. The bacterial cell dies and bursts open releasing the newly formed bacteriophages. These then seek other bacterial host cells to infect.

As this cycle continues, bacteriophages can create an “epidemic” and quickly destroy populations of target bacteria. Beneficial bacteriophages have been used for many purposes, including as therapeutic agents prior to the advent of antibiotics. More recently they have been used in packaged cold cut meats as a United States Food and Drug Administration (FDA) “generally recognized as safe” additive to control pathogenic bacteria such as Listeria monocytogenes.

Jiang and her students have explored the use of bacteriophages as a low-cost and effective method to control Salmonella in rendering plant environments. The team first identified possibly sources of Salmonella contamination within rendering processing environments. Next they isolated bacteriophages specific to Salmonella then used these bacteriophages to reduce Salmonella attachment and biofilm formation on surfaces.

In the first part of the study, the research team analyzed 108 samples collected from potential sites for Salmonella, such as rendering plant raw material receiving areas, crax grinding, and at finished meal load-out. Of the collected samples, 73 percent were positive for Salmonella. There were 16 serotypes, with Salmonella Typhimurium and Salmonella Mbandaka the dominant ones. Additionally, 10 Salmonella serotypes were judged to be strong biofilm formers, meaning they have the ability to create a protective environment for themselves on surfaces. As expected, the raw material receiving areas were the primary source of Salmonella but the surfaces surrounding crax grinding and the finished meal load-out areas harbored Salmonella in biofilms. It was also noted that the same serotypes of Salmonella were found in both raw materials and in the finished meal load-out areas suggesting potential cross-contamination had occurred between different areas of the rendering processing environment.

The team collected samples from within rendering plants and isolated bacteriophages capable of infecting/killing Salmonella serotypes found to be present in rendering processing environments. They were successful in isolating 94 bacteriophages against Salmonella. From these, they selected the best for destroying the 10 serotypes of biofilm-forming Salmonella they had noted in the previous experiment, creating a “cocktail” of multiple types of bacteriophages to develop a potent Salmonella-killing product. They studied how to optimize the killing power of the bacteriophages and how to use these as a supplement along with sodium hypochlorite (bleach) for use in boot baths.

Boot baths are designed to reduce cross-contamination from different areas of processing plants. Workers scrub their boots thoroughly on each trip through the boot bath. However, boot baths have several inherent problems. One is that typical boot bath disinfectants can lose effectiveness with time and soil deposition. Two, persuading workers to regularly use boot baths properly is difficult. Many times workers will simply step over or around the bath, completely avoiding contact with the disinfectant solution. Other times, workers often step only one foot into the bath and then keep walking.

After preliminary testing of the Salmonella-specific bacteriophage cocktail on different surfaces typical in rendering environments, Jiang and Gong moved their work to rendering plants in an attempt to reduce Salmonella contamination on workers’ boots. They put their bacteriophage cocktails in the boot bath and observed that Salmonella populations on workers’ boots were reduced by 84.5 percent. They combined their bacteriophage cocktail with diluted sodium hypochlorite and the Salmonella populations on boots were reduced by 92.9 percent. This mixture produced an added benefit of extending the effective time of the solution.

Normally when sodium hypochlorite is used in boot baths, it loses its effectiveness to kill bacteria over time due to soil contaminants from the boots binding with it along with off-gassing of the chlorine. The loss of hypochlorite solution strength also is affected by heat and sunlight. Addition of the bacteriophage cocktail to the solution allowed improved bactericidal activity. Finally, the researchers combined
bacteriophage treatment with daily boot scrubbing and noted a 93.2 percent reduction in Salmonella populations on workers’ boots.

Use of bacteriophages to reduce Salmonella biofilms could be beneficial to rendering quality control programs and help prevent cross-contamination. Bacteriophages are effective and non-corrosive. An added benefit is that, upon killing their target bacterium, they multiply and release many copies of themselves seeking out other Salmonella bacteria to destroy. This “epidemic” likely will create a protection within a rendering plant to help fight Salmonella using an environmentally friendly biological control method. Use of this technology will further improve worker safety and produce high-quality, Salmonella-free rendered animal meals.

Gong completed his PhD degree in May 2016 and now works for a rendering company in the Midwest.

October 2016 RENDER | back